The overall goal of this proposal is to define the function of glutathione (GSH) in mammals. Mice deficient in the heavy (catalytic) subunit of gamma-glutamyl cysteine synthetase (gamma-GCS-HSU), the rate limiting enzyme in GSH synthesis, diet at post coital day 7.5 and completely lack GSH; however, cell lines derived from gamma-GCS- HSU-deficient embryos survive if GSH is provided in the medium. These cells contain only 50-60 mum GSH (1-2% of wild type levels). Preliminary data indicate that removal of GSH from the medium produces a fall in GSH to undetectable levels, increased hydroxyl radical formation, activation of JNK/SAPK and cell death. These data suggest that very low GSH levels (,1% of wild type cells) whether "spontaneous" or induced by chemicals/radiation may be a key trigger that initiates the oxidative stress response and apoptosis. The gamma-GCS-HSU-deficient mice and cells we have developed and additional knock-out mice and cells we plan to construct provide a unique opportunity to test four hypotheses about GSH function: 1. Only low levels of GSH are required for survival of unstressed cells, but high levels are necessary to protect cells from chemical and radiation injury, and this protection is directly proportional to GSH level. 2. Very low/absent levels of cellular GSH activate a cellular stress response program that includes JNK/SAPK, p38 MAPK and NFkappaB, and activation of these pathways by chemicals radiation depends upon their ability to lower GSH levels. 3.Very low-absent levels of cellular GSH potentiate apoptotic cell death, and chemicals/radiation that induce apoptosis do so by lowering cellular GSH levels. The process involves suppression of anti-apoptotic factors and activation of pro-apoptotic factors. 4. Only low levels of GSH are needed for development and survival of unstressed mice. These low levels of GSH render mice hypersensitive to chemical injury.